From the Department of Cardiovascular Pharmacology, SmithKline Beecham
Pharmaceuticals, King of Prussia, Pa.
Correspondence to Xinkang Wang, PhD, Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, PO Box 1539, UW2511, King of Prussia, PA 19406. E-mail Xinkang_Wang-1{at}SBPHRD.COM
Background and Purpose—Differential gene expression has been
reported following the onset of focal stroke. To identify de novo
expression of ischemia-induced genes, we applied subtractive
cDNA library strategy to identify the genes that are selectively
upregulated by focal stroke.
Methods—Spontaneously hypertensive rats were subjected to
permanent occlusion of the middle cerebral artery (MCAO). mRNAs
prepared from ischemic and nonischemic cortex 2 and 12
hours after MCAO were subtracted, and a subtractive cDNA library was
constructed. A cDNA that encodes for tissue inhibitor of
matrix metalloproteinase-1 (TIMP-1) was identified in the subtractive
cDNA library. The temporal expression of cortical TIMP-1 mRNA was
further characterized in ischemic cortex subjected to permanent
or temporary (160-minute) MCAO.
Results—A panel of genes isolated from the subtractive cDNA
library was subjected to Southern analysis to confirm
ischemia-induced gene expression. TIMP-1 demonstrated robust
induction after ischemic injury. Time-course studies revealed
that TIMP-1 mRNA was induced threefold over controls at 12 hours
(P<.001, n=4 animals) and reached a peak level at 2
days after permanent MCAO (sevenfold increase, P<.001).
Similar induction profile of TIMP-1 mRNA was observed in the
ischemic cortex after temporary MCAO followed by
reperfusion.
Conclusions—This work demonstrated the utility of
subtractive cDNA library strategy for discovery of genes differentially
expressed in focal stroke. Furthermore, our data implicate TIMP-1 in
ischemia-induced brain injury.
Department
of Neurology and Neuroscience,
Johns Hopkins University School of Medicine,
Baltimore, Maryland
© 1998 American Heart Association, Inc.
Original Contributions
Subtractive Cloning Identifies Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) Increased Gene Expression Following Focal Stroke
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